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88 The Open Dentistry Journal, 2013, 7, 88-93

1874-2106/13 2013 Bentham Open
Open Access
CRP and IL-1B Gene Polymorphisms and CRP in Blood in Periodontal
Disease
Auerkari EI
1,2,*
, Suhartono AW
1
, Djamal NZ
1
, Verisqa F
1
, Suryandari DA
3
, Kusdhany LS
2,4
,
Masulili SLC
5
and Talbot C
6

1
Department of Oral Biology, Faculty of Dentistry, University of Indonesia, Jakarta Indonesia
2
Centre for Ageing Studies, University of Indonesia, Jakarta Indonesia
3
Department of Medical Biology, Faculty of Medicine, University of Indonesia, Jakarta Indonesia
4
Department of Prosthodontics, Faculty of Dentistry, University of Indonesia, Jakarta Indonesia
5
Department of Periodontology, Faculty of Dentistry, Universitas Indonesia, Jakarta, Indonesia
6
Department of Genetics, University of Leicester, UK
Abstract: Recent studies have suggested an association between periodontal disease (PD) and the systemic polygenic dis-
eases such as cardiovascular disease (CVD). These are thought to be associated because of interrelated environmental,
epigenetic, and genetic risk factors. The involved candidate genes include the IL-1B gene, encoding the pro-inflammatory
cytokine IL-1!, and the CRP gene encoding the C-reactive protein (CRP), also a known marker of inflammation. How-
ever, as the details are not well known on the genetic variation influencing the risk factors, this work aimed to evaluate the
distribution of selected polymorphisms of IL-1B and CRP genes, and serum CRP level, in comparison with the PD status.
For this purpose, periodontal health was assessed, serum CRP levels measured and polymorphism status of IL-1B and
CRP genes determined from samples of peripheral blood taken from 101 consenting Indonesian adult males. The results
show that severe PD was significantly associated with age and smoking, as expected, but not with the polymorphisms of
IL-1B or CRP (1444). However, a significantly lower fraction of subjects with normal periodontal health than subjects
with PD showed the heterozygous type polymorphism of CRP (717). There was no significant difference in the fraction of
cases with elevated serum CRP level between subjects with normal health and those with PD, and further study with a
larger sample is recommended. The observed association between polymorphism of CRP (717) and periodontal health is
suggested as a complementary indicator of the risk to PD for the Indonesian male population.
Keywords: Periodontal disease, cardiovascular disease, IL-1B, C-reactive protein, polymorphism, serum level.
INTRODUCTION
Periodontitis is a complex multifactorial disease, usually
initiating late and progressing slowly to a chronic phenotype
or severe periodontal disease (PD) that is promoted both by
environmental factors like smoking, pathogenic bacteria and
stress, and by genetic factors [1]. The pathogenesis of PD
involves repeated attack of the subgingival bacteria and the
host immune defences that result in an inflammatory process
contributing to the periodontal tissue destruction. The perio-
dontal structure is a site of unusual opportunity for pathogen
attack with the non-shedding tooth surface allowing forma-
tion of persistent bacterial biofilms that can maintain prox-
imity to the periodontal tissue [2, 3]. The initial and reversi-
ble gingivitis may develop to periodontitis with the perio-
dontal ligament detaching from cementum to form


*Address correspondence to this author at the Department of Oral Biology,
Faculty of Dentistry, University of Indonesia, Jakarta, Indonesia;
Tel: +62213910344; Fax: +622131906289;
E-mail: eauerkari@yahoo.com, elza.ibrahim@ui.ac.id
periodontal pockets, and the damage extends further to cause
alveolar bone resorption, gingival recession, tooth mobility
and finally tooth loss. Of the over 300 bacterial species iso-
lated from dental plaque, common Gram-negative species of
the periodontal pockets include Aggregatibacter (previously
Actinobacillus) actinomycetemcomitans, Porphyromonas
gingivalis and Tannerella (previously Bacteroides) for-
sythus. However, while the bacterial interactions or bacterial
plaque are required for the inflammatory condition, they do
not explain all cases, for example when periodontitis is ob-
served with little bacterial plaque, or no periodontitis in spite
of heavy plaque. The progression and severity of the disease
is affected by the strength of the host immune defences and
other responses, and the variation in the susceptibility im-
plies an effect of genetic factors [3, 4]. The multifactorial
nature of the disease suggests multiple gene associations
with a modest or weak individual influence that combined
together with other factors have a significant effect on the
variation of the manifested outcome [5]. Also other multifac-
torial diseases like cardiovascular disease (CVD) similarly
involve chronic inflammation. CVD is caused by atheroscle-
CRP and IL-1B Gene Polymorphisms and CRP in Blood The Open Dentistry Journal, 2013, Volume 7 89
rosis (hardening) of blood vessels, the process through which
deposit of fatty substances, cholesterol, cellular waste prod-
ucts, calcium and other substances build up in the inner arte-
rial lining. The atherosclerotic plaque will cause symptoms
and complications as a result of narrowing of the arterial
lumen and narrowing of blood flow to the heart [6]. Epide-
miological studies suggest an association between CVD and
PD [2, 7, 8]. Suggested mechanisms that can result in such
an association include common risk factors including smok-
ing and infections that promote release of proinflammatory
cytokines like interleukine-1 or IL-1, and genetic factors
related to the regulatory networks of the inflammatory
mechanisms [7, 9].
One sensitive marker of inflammation is C-reactive pro-
tein (CRP) that is produced particularly in the acute phase of
inflammation, infection and trauma in response to pro-
inflammatory cytokines like IL-1 and IL-6 [2]. Single nu-
cleotide polymorphisms (SNP) of IL-1B gene, located on
chromosome 2q13-q21 and which encode the IL-1! cytokine
protein, have been reported to be associated with the baseline
blood CRP levels of healthy individuals, and it has been
suggested that IL-1B regulates the basal CRP levels [3].
Considering the documented evidence on the association
between CVD and PD, with expected common genetic risk
factors, it is of interest to explore the mechanisms of the as-
sociation in further detail. The details can be expected to
involve the IL-1B gene as one of the primary candidates, and
possibly also the CRP gene that is expressed as C-reactive
protein in the affected tissue, and in blood. The impact of the
genetic variation of particular interest can be assessed by
evaluating the polymorphism status in these genes within the
population, which in the present case is Indonesian, with
respect to the corresponding status of PD. With more de-
tailed information on the mechanisms involved one can hope
for improved predictors of risk to CVD, PD, and other sys-
temic chronic disease, and possibly even for routes towards
improved therapies.
This work aimed to evaluate the distribution of selected
polymorphisms of IL-1B and CRP genes, and serum CRP
level with respect to the PD status in a sample from the In-
donesian population. Implications were also to be considered
on predicting the risk to PD, based on the potential associa-
tions between the patterns of polymorphisms, serum CRP
level, and the severity of PD.
MATERIALS AND METHODOLOGY
Subject Recruitment, Classification, and Measurement of
CRP Level
In total, 101 consenting Indonesian male adults were in-
cluded in this study, with an age range of 25-65 years and
median of 39.8 years. The ethical clearance for the work was
granted by the Ethical Committee of the Faculty of Den-
tistry, University of Indonesia and all patients signed written
informed consent. Intra oral examination and periodontal
health status (plaque index, papillae bleeding index, attach-
ment loss (AL), probing depth) were assessed for all subjects
using a standardized procedure at six location on each tooth.
For severity of PD, the subjects were divided into three
groups: mild / healthy (AL ! 2 mm); moderate (AL > 2-4
mm) and severe (AL > 4 mm) PD. Examination of clinical
attachment loss (CAL) was measured at six tooth surfaces of
all teeth. Also, levels of CRP and alkaline phosphatase were
measured from peripheral blood samples by using Immuno-
turbidimetric technique and ALP IFCC Gen2 .
DNA Isolation, PCR Amplification and RFLP Genotyp-
ing
To survey the genotype-phenotype variations related to
the gene locus polymorphisms of IL-1B and CRP, the poly-
morphism status of these genes was determined from sam-
ples of peripheral blood.
For isolation of DNA, 3 mL of peripheral blood was
taken from each of the 101 subjects, placed in 15 mL tubes
containing 9 mL of red blood lysis solution (1.45M NH
4
Cl,
5mM anhydrous EDTA, and 0.1M KHCO
3
) and incubated at
room temperature for 10 min. The sample was then centri-
fuged at 1500 rpm for 10 min at room temperature, and the
supernatant was removed to leave a precipitate of mononu-
clear leukocytes. These steps were repeated to obtain a white
pellet and a supernatant containing no red blood cells. To
this pellet 2 mL of cell lysis solution was added and pipetted
until homogeneous, and incubated in a water bath at 37C
for 30-60 min until completely homogeneous. Then 1.3 mL
of protein precipitation solution (5M ammonium acetate)
was added, vortex mixed for 15-20 s and centrifuged at 3000
rpm for 15 min at 4C, producing a light brown precipitate
(proteins) and the supernatant containing DNA. The super-
natant was poured into a new Falcon tube with 2.3 ml of cold
isopropanol. The tube was inverted up to 20-30 times until
showing a collection of DNA strands. The supernatant was
removed and 1.3 mL of 70% ethanol was added for washing,
and the DNA solution was centrifuged at 3000 rpm for 5 min
at 4C. After discarding supernatant, the DNA was dried in
open air by reversing the tube, then DNA was rehydrated
with a solution of 200-300 uL TE (Tris-HCl EDTA) and
incubated in a water bath at 37C for 2 h. The solution was
transferred into 1.5 mL sterile microcentrifuge tubes and
stored at -20C until further examination.
PCR amplification of DNA fragments was carried out by
using Perkin Elmer GeneAmp PCR System 9700 with
PCR Master Mix, BiomixRed (Bioline). The DNA samples
were amplified in 35 cycles of an initial denaturation at 94C
for 5 min and cycles of denaturation, annealing and elonga-
tion (primers and PCR optimization are shown in Table 1).
The end of the cycle included an extension at 72C for 7
min. After completion the amplicons were stored at 4C.
The polymorphisms of IL-1B, CRP717 and CRP1444
were analyzed with PCR-RFLP using the restriction en-
zymes TaqI, SacII and Bsp, respectively, for cutting at the
sites of polymorphisms [3, 5]. For each amplicon to be cut,
the corresponding restriction enzyme was added into 10 L
of the amplified DNA fragments, 2 L buffer solution and 18
L ddH
2
O, incubated in water bath at 37C for 3 h and fi-
nally at 65 C for 20 min for enzyme inactivation. For frag-
ment separation, 5 L of the resulting product was mixed
with 2 L of tracking dye (0.25% bromophenol blue, 0.25%
xylene xyanole, 25% sucrose) and subjected to electrophore-
sis on 3% agarose gel (Promega) containing 1 L ethidium
bromide (0.5 mg/mL) in 1X TAE buffer solution (0.04 M
90 The Open Dentistry Journal, 2013, Volume 7 Auerkari et al.
Tris-acetate, 0.002 M EDTA pH 8.0) at 90 V for 60 min.
Using 100 bp DNA ladder markers (Promega), the results
were inspected under UV illumination (UV Filter TM Spec-
troline) and recorded with a digital camera (Fig. 1).
Statistical Analysis
Chi-square testing with SPSS 18.0 was mainly used in
the statistical analysis, both for comparing results in the test
groups and for assessing the allele and genotype frequencies
with predictions with respect to the Hardy-Weinberg equilib-
rium. Statistical significance was assumed with p < 0.05.
RESULTS
The results are summarized in Table 2, where they are di-
vided into groups of normal, moderate and severe periodontal
disease. In this classification the normal cases included the
lowest number of cases (11). The mean age was highest for
the severe PD group, and for all groups SD is similar and also
about the same as the difference between the means of the
normal and severe PD groups. Of the subjects with severe PD,
many fewer were non-smokers (28.6%) than smokers
(71.4%). In contrast, 41.7% of subjects with moderate PD
were non-smokers, and only 10.4% of the subjects in this
group (38.1% in the severe PD group) were heavy smokers
with lifetime exposure to at least 100,000 cigarettes. In the
normal group, 63.6% of the subjects were non-smokers and
none were heavy smokers. The median and range of lifetime
exposure to smoking showed a systematic increase from nor-
mal periodontal health to moderate and severe PD groups (Fig.
2). The measured range in the serum levels of C-reactive pro-
tein varied between 0.1 and about 20 mg/L.
The most common type of IL-1B polymorphism was the
one with common homozygotes (CC), at a frequency of
more than 90% for all groups of PD severity. For the het-
erozygotes type (CT), three cases (7.1%) only appeared in
the severe PD group, and for rare homozygotes (TT) only
two cases (4.2%) in the moderate PD group. For CRP (717)
polymorphism, the common homozygotes (SS) occurred at
frequencies of 54.2%/59.5% in the moderate/severe PD
groups, but at 90.9% frequency in the normal group. The
heterozygotes (Ss) appeared at frequencies of 37.5% and
33.3% in the moderate and severe PD groups, respectively,
but not at all in the normal group. The rare homozygotes (ss)
were observed at fairly similar though slightly decreasing
rates of 9.1%, 8.3%, and 7.2% in the normal, moderate and
severe PD groups, respectively. For CRP (1444) polymor-
phism, the more common homozygotes (BB) appeared only
at low and decreasing frequencies of 9.1%, 6.25% and 4.8%
in the same grouping order, while the heterozygotes (Bb)
occurred at a frequency of 90% or higher in all groups, but
rare homozygotes (bb) not at all.
DISCUSSION
In this work the polymorphisms of IL-1B (3954) and
CRP (717, 1444) genes have been compared to the periodon-
tal health and serum CRP level of 101 Indonesian male sub-
jects. This sample included relatively few normal (healthy)
subjects in comparison with the number of those with mod-
erate or severe PD; this largely reflects the unfortunately
high prevalence of PD in the Indonesian population. The
results show a clear and expected relationship between PD
severity and smoking habit, so that severe PD was signifi-
cantly associated with higher lifetime smoking exposure, and
normal oral condition and moderate PD with non-smokers.
Note however that there are clearly other significant contrib-
uting factors than smoking (e.g. individual performance levels
Table 1. Primers and PCR Cycles for Amplifying the Target Genes
Gene Primers PCR cycles
IL-1B 5- CTCAGGTGTCCTCGAAGAAATCAAA-3'
5- GCTTTTTTGCTGTGAGTCCCG-3'
35 cycles, 94C(30"), 58C(30"), 72C(30")
CRP717 5'-ACTGGACTTTTACTGTCAGGGC -3'
5'-ATCCCATCTATGAGTGAGAACC-3'
35 cycles, 94C(30"), 58C(30"), 72C(30")
CRP1444 5"-AGCTCGTTAACTATGCTGGGGCA-3"
5"-CTTCTCAGCTCTTGCCTTATGAGT-3"
30 cycles 94C(30"), 62C(30"), 72C(30")


a) b) c)
Fig. (1). Fragment separation in electrophoresis to show polymorphisms of a) IL-1B; b) CRP (717) and c) CRP (1444).
CRP and IL-1B Gene Polymorphisms and CRP in Blood The Open Dentistry Journal, 2013, Volume 7 91
Table 2. Characteristics of the Study Population in Terms of Severity of Periodontal Disease; Age and CRP level as mean SD; LE =
Lifetime Exposure as Number of Cigarettes; NS = not Significant
Variable Normal Moderate PD Severe PD P
N 11 48 42 -
Age (years) 35.0 9.6 37.2 9.3 44.2 9.2 0.001
a)
Smoking:
Non-smoker
LE < 100000
LE # 100000

7 (63.6%)
4 (36.4%)
0 (0%)

20 (41.7%)
23 (47.9%)
5 (10.4%)

12 (28.6%)
14 (33.3%)
16 (38.1%)

0.005
CRP (mg/L)
N with > 3 mg/L
2.1 3.9
1 (9.1%)
2.0 2.3
8 (17.0%)
2.5 3.9
8 (20.0%)
0.743 NS
a)

0.491 NS
IL-1B (3954):
CC
CT
TT

11 (100%)
0 (0%)
0 (0%)

46 (95.8%)
0 (0%)
2 (4.2%)

39 (92.9%)
3 (7.1%)
0 (0%)

0.587 NS
CRP (717):
SS
Ss
ss

10 (90.9%)
0 (0%)
1 (9.1%)

26 (54.2%)
18 (37.5%)
4 (8.3%)

25 (59.5%)
14 (33.3%)
3 (7.2%)

0.047
CRP (1444):
BB
Bb
bb

1 (9.1%)
10 (90.9%)
0 (0%)

3 (6.25%)
45 (93.75%)
0 (0%)

2 (4.8%)
40 (95.2%)
0 (0%)

0.857 NS
a)
Kruskal-Wallis test

Fig. (2). Impact of the estimated number of smoked cigarettes during lifetime for subject groups of normal periodontal health, moderate PD
and severe PD (asterisks refer to outlier cases).

of oral hygiene and immune response), as is seen from the
increasing scatter in the lifetime exposure with the severity
of PD in Fig. (2). Also an expected association with age was
seen, so that the mean age of the subjects with severe PD
was higher than for those with normal oral health and mod-
erate PD. However, the grouping of normal condition, mod-
erate PD and severe PD was not significantly associated with
the polymorphisms of IL-1B or CRP (1444) in the sample

92 The Open Dentistry Journal, 2013, Volume 7 Auerkari et al.
population. The only exception was CRP (717) where the
heterogeneous type in normal subjects occurred at a signifi-
cantly lower fraction of cases than in subjects with PD; even
here, no significant difference was seen between subjects
with moderate and severe PD. The levels of serum CRP were
varying strongly in all groups of PD severity, but the fraction
of subjects with serum CRP level above 3 mg/L was lower
for cases of normal periodontal health than for cases of mod-
erate or severe PD. A large scatter is not unexpected since
inflammation other than that related to PD can have much
influence on the serum CRP levels.
The pro-inflammatory IL-1B can be blocked with the IL-
1 receptor antagonist IL-1Ra (anakinra) that has been ap-
proved for treatment of some chronic autoinflammatory dis-
eases like rheumatoid arthritis [8]. Until recently the IL-1
family only consisted of two main members (IL-1A and IL-
1B) but has considerably expanded to include at least 11
members of which some like IL-18, IL-36A, IL-36B, and IL-
36G are also pro-inflammatory factors, and some others ap-
pear antagonist or anti-inflammatory (IL-1Ra, IL-36Ra, IL-
37) or with unknown function (IL-38). With only partially
known receptors and coreceptors, their overall roles remain
to be described in detail but are likely to be fairly complex
judging from the known signalling networks related of IL-1B
[9]. The observed frequencies of IL-1B (3954) genotype CC
(Table 2) were clearly higher (more than 90%) in this work
for Indonesian male adults than what has been reported pre-
viously for European subjects [15].
Chronic periodontitis typically involves a persistent in-
fection of the periodontal pocket by Gram-negative bacteria
like A. actinomycetemcomitans. This micro-organism pro-
duces a leucotoxin that induces degranulation and lysis in
human neutrophils, caspase-1 activation, and abundant secre-
tion of IL-1! from human macrophages, promoting tissue
destruction including loss of alveolar bone [9,10]. Therefore
the periodontal induction of IL-1B activity by the bacterial
leucotoxin largely explains tooth loss in advanced PD. With
local periodontal inflammation mediated by IL-1! that also
enters circulation, the observed association is perhaps to be
expected with CVD, another chronic systemic disease pro-
moted by pro-inflammatory cytokines [2,9,11,14]. Neverthe-
less, the reported association between PD and CVD, and
associations with them and individual polymorphisms of IL-
1 and CRP genes, can be diluted by the complex polygenic
nature of the overall regulatory networks with a relatively
small contribution by any single gene. If the associations
between individual genes and their polymorphisms are rela-
tively weak, this also means that the individual polymor-
phisms alone are not particularly powerful risk indicators to
such disease. While the conventional indicators may remain
more useful, like the directly measured extent of pocket
depth and alveolar bone loss for PD, and high blood pres-
sure, smoking, obesity, and CRP level for CVD, successful
treatment of PD can reduce inflammation and circulating IL-
1 and CRP levels, with potentially reduced risk to CVD
[12,13,16,17]. Also, potential additional indicators like CRP
(717) polymorphism status may serve as a complementary
factor to the current array of standard indicators, at least if it
can be in future more conveniently tested.
CONCLUSION
A significantly lower fraction of subjects with normal
periodontal health than those with PD showed the heteroge-
neous type polymorphism of CRP (717) that hence may pro-
vide a complementary indicator of the risk to PD. In contrast,
no significant association was found between periodontal
health and the polymorphisms of IL-1B or CRP (1444) in the
Indonesian male sample population. However, considering
the currently available evidence [16-18], the association be-
tween PD and CVD appears to remain sufficient to justify
informing patients with severe PD that they may have an
increased risk of CVD.
As the number of the subjects with a normal periodontal
health in this work was relatively small, confirmation with
further study of a larger sample population is necessary.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flicts of interest.
ACKNOWLEDGEMENTS
The authors wish to gratefully acknowledge the financial
support from the University of Indonesia (DRPM-UI 2010)
and the Ministry of Education and Culture of the Republic of
Indonesia.
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Received: March 20, 2013 Revised: March 27, 2013 Accepted: March 27, 2013
Auerkari et al.; Licensee Bentham Open.
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